This invention provides new methods of selecting a binding protein with enhanced binding affinity for a binding partner relative to a reference binding protein. In particular, the invention provides methods of selecting antibodies with enhanced affinity for an antigen relative to a reference antibody. This process, “affinity maturation”, thereby provides binding proteins, e.g., antibodies, with superior binding capabilities.
Antigen-specific antibodies can be produced by a variety of methods including hybridoma technology (e.g., Kohler & Milstein, Nature 256:495-497, 1975) or selection in vitro using phage or yeast display libraries (e.g., Hoogenboom et al., Immunotechnology 4: 1-20, 1998; Boder & Wittrup, Methods Enzymol 328:430-44, 2000). However, antibodies derived from these methods often have sub-optimal binding affinities. Affinity discrimination among nonspecific antibodies in vitro may be accomplished by equilibrating a mutagenic library of the antibody in question with soluble cognate antigen under conditions in which the concentrations of both antibody and antigen are maintained below the target equilibrium dissociation constant. Competition must be avoided to prevent abundant low-affinity variants from excluding rarer high-affinity variants. Operationally, there are two additional requirements: (1) the antibody library must be displayed by a vehicle such as a bacteriophage or a cell, which couples the antibody to its coding sequence, and (2) the antigen must be coupled to a tag which allows quantitative separation of antigen-bound antibody from unbound antibody. The main drawback of these procedures is that for most applications, affinities in the nanomolar Kd range are desired and these antibodies are sufficiently rare to be easily lost at sub-nanomolar working concentrations. Such difficulties have led to selecting for lower dissociation rate constants (kd, off-rates) to improve antibody affinities. Selection for lower off-rates is usually performed under saturating conditions where antigen-antibody complexes remaining intact after a time proportional to the inverse of the target off-rate can be recovered separately from dissociated antibodies. However, in these procedures it is frequently desired that off-rates for antibodies are in the range of 1×10−4 sec−1, which corresponds to a half-life of ˜2 hours. Many antibodies and especially antigens undergo significant irreversible denaturation in vitro on such time scales at ambient or physiological temperatures. Also, off-rate selection in the absence of on-rate selection (lower ka) tends to bias the selection toward variants that refold into stable complexes and therefore tend to disfavor increased on-rates.
Thus, there is a need to develop new methods for obtaining binding molecules, e.g, antibodies, that have improved binding affinities for their binding partners. The current invention fulfills this need. Further, for many antibody applications, a successful protocol for affinity maturation in vitro will be one that produces improvements in both on-rate and off-rate while maintaining or increasing the specificity for the intended antigen. The methods described herein also provide these improvements.